Chemical Composition of Citrus Fruits (Orange, Lemon, and Grapefruit...
8 downloads
129 Views
1MB Size
Chapter 3
Chemical Composition of Citrus Fruits (Orange, Lemon, and Grapefruit) with Respect to Quality Control of Juice Products Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
1
1
2
Agnes Sass-Kiss , Marianna Toth-Markus , and Miklos Sass 1
Department of Analytical Chemistry, Central Food Research Institute, Herman Otto 15, 1022 Budapest, Hungary Department of Zoology, Eötvös Lorand University of Sciences, Budapest, Hungary 2
With expanded consumption and production of citrus fruits and increased demand for healthy nutrition comes the need for knowledge of the chemical constituent of citrus fruits and their products. Thus, the compounds of citrus fruits were analysed. Constituents that are characteristic for citrus species or different fruit parts are useful to determine the authenticity and detect the adulteration in quality control of citrus juices. In expectation of the high profit, citrus juices are subject to falsification because they can be easy manipulated. Many forms of adulteration have been found from simple dilution with water or substitution of cheap ingredients (sugar, acid, colorant essence, and other types of cheap fruit juices) to sophisticated methods as addition of by-products (peel extract or pulp wash) to the juice. Fruit composition is influenced by a large number of natural factors, namely the variety of fruit, the geographical location, the climatic zone, the soil, the degree of maturity as well as other factors. The composition of juice products may be influenced by technology used in processing and packaging.
In spite of all different variables, experiences have shown that a large number of parameters and values are subject to statistical laws. These parameters can be used for the evaluation of juice products with respect to their quality, authenticity and identity, in spite of remarkable variations in their contents. © 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
25 It is not possible to establish universally applicable standards for authenticity of processed fruit juices but it is possible to provide minimum or maximum values and/or ranges for individual constituents. The code of practice of Association of Industry of Juices and Nectars (A.I.J.N.) from Juices and Vegetables of the European Union includes reference guidelines, which represent a collection of such minimum and maximum values and/or ranges. These values are used in our laboratory for evaluation the quality of juice products available in the market. Table 1 shows the results of some selected orange juice products purchased from the Hungarian market in 1997, 1999 and 2000. The presence of high concentration of water soluble pectin, high hesperidin and calcium may indicate the addition of peel extract and pulp wash. The low concentrations of L-malic acid and isocitric acid, and as a consequence, high ratio of citric acid to D-isocitric acid are also signs of manipulation. As an effect offrequentcontrol, the quality of 100 % orange juice products present in the Hungarian market has improved since 1997 - Out of 12 samples examined, one product had about 50 % fruit content and only one was perfect. In 1999, out of 8 samples two had a pulp wash addition, while in 2000from10 samples studied, only one was really manipulated. The compounds mentioned above should be used together to evaluate the manipulation of citrus juice products. None of these compounds are specific enough to characterise the different part of citrus fruits as juice, albedo orflavedoand the various citrus species. Some early report discussed that there were some differences in protein composition of the exo-, mesoand endocarp of orange, grapefruit, lemon and in species specific immunogenicity of orange and lemon juice. According to these descriptions, proteins seem to be specific enough and particularly relevant to characterise the species and the parts of citrus fruits. The goal of our program was to develop a new and modern immunochemical method for quality control of commercial citrus juices to detect the manipulation with peel extract. The experiments consist(ed) of six steps. 1. Identification of juice, albedo, flavedo specific peptides in various citrus species by analytical SDS-PAGE 2. Isolation of these characteristic peptides by preparative gel electrophoresis 3. Development of antibodies against the isolated peptides 4. Analysis of the specificity of isolated peptides on Western blot. 5. Qualitative analysis to detect the peel and juice peptides in citrus juice products by Western blot. 6. Determination of the concentration of characteristic peptides in juice products by ELISA in the near future. We investigated orange (Citrus sinensis), lemon (Citrus limon), grapefruit (Citrus paradisi).
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Table I. Analysis of 100% orange juices from the market
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
27
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
28 Sample preparation. The fruits were peeled. The albedo and flavedo or peel (albedo+flavedo) were separated and cut into small pieces. After peeling the fruits, the juice was obtained by afruitjuice-maker. One gram of peel was extracted in 10 ml water (10 % peel extract). The juice and peel extract were filtered through filter paper. The filtrate was centrifuged at 15,000 xg for 20 min. The sediment was resuspended in Tris-HCl (pH 8.3) buffer and mixed with equal volume of Laemmli's sample buffer. The solution was boiled for 3 min. After centrifugation at 15,000 xg for 10min the supernatant was used for SDS-PAGE. Gel-electrophoresis. The SDS-PAGE was carried out according to Laemmli (1) on a 13.6% (w/v) separation gel overlaid by 3% stacking gel in a BioRad Mini Gel Chamber. Gels were stained by Coomassie BBR. Separation of peptides by preparative gel-electrophoresis. Preparative SDS-polyacrylamide gels (1.5mm thick) were used to separate the peptides of the sample and the standard respectively. After electrophoresis, two edges of the gel were cut and stained to determine the position of the peptides of interest, which were then cut outfromthe gels. Peptides were electroeluted into dialysis bags (Cellulose membrane retainings proteins with MW 12,400 Da) in a Mini Trans Blot chamber (BioRad) for 600 V*h. The peptide samples (~ 6 ml) were dialyzed overnight against distilled water at 4°C and lyophilised. The peptides were redissolved in 60-100 μΐ offinalvolume. The purity of peptides was tested by analytical SDS-PAGE and the procedure was repeated again to obtain electrophoretically homogeneous samples. Preparation of antibodies. The peptide (in 100 μΐ of PBS with equal volume of Freund's complete adjuvant) was injected into the skin of male BALB/c mice. Animals were boosted 3 weeks later with the same amount of protein. The titer of antibodies was tested in blood samples collected from the tip of tail of the mice. The third booster was normally enough to achieve a useful titer of antibodies against our peptides. After completing the immunizations animals were bled and the IgGfractionwas separated from the sera by ammonium sulfate precipitation. Immunoblotting. Peptides were transferred from SDS-polyacrylamide gel to a sheet of nitrocellulose filter (BioRad, 0.2 μπι pore size) by electroblotting according to the method of Towbin et al (2). The nonspecific binding sites were blocked by 5 % Carnation non fat dry milk powder in TBS buffer (0.15 M Tris-HCl, 0.5 M NaCl pH 7.0). After washing three times with TTBS buffer (TBS buffer containing 0.05 % Tween 20) the nitrocellulose sheet was incubated in the presence of the first antibody, diluted in TBS for 12 h at 4 °C. Blot was washed three times in TTBS and incubated with the second antibody (alkaline phosphatase labelled anti-mouse antibody, BioRad) dissolved in TBS in 1:1000 for one hour at room temperature. After the final washes, the blot was developed in a solution of freshly prepared substrate (BCIP and NBT, Bio-Rad). Molecular masses of the positive bands were determined using BioRad prestained standards (Myosin 206 kDa, β-galactosidase 120 kDa, Bovin serum albumin 84 kDa, Ovalbumin 52 kDa, Carbonic anhydrase 36 kDa, Soybean tripsin inhibitor 30 kDa, Lysozyme 22 kD, Aprotinin 7,5 kDa).
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
29
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
Juice and peel specific peptides In citrus fruits, several peptides were identified that occurred exclusively in the juice, or the flavedo or the albedo (3). In orange, seven peptides could be found exclusively in the juice, two of them appearing exclusively in the flavedo. Only in albedo one very pale peptide was detected, which did not appear in the flavedo or in the juice. In grapefruit, seven peptides in the juice and three peptides in the flavedo and one very pale peptide band in the albedo were identified as specific ones, which were not present in the other two parts of the fruit. In lemon juice three, in the albedo one, in the flavedo two specific peptides appeared that did not occur in other parts of the fruit. The molecular mass of isolated peptides are shown in Table II. The number of peptides isolated by preparative gel electrophoresis were two from orange 2,fromthree grapefruit juice, onefromgrapefruit peel and two from lemon juice. The tissue and species-specific distribution of the antigens have been studied in grapefruit, lemon, and orange by Western blots. Table III shows the distribution of isolated peptides in the juice and peel samples of citrus fruits. The 27 kDa peptide and the 82 kDa peptide occurred in the juice of all citrus species studied, but did not appear in their peel samples (the antibody developed against 27 kDa peptide isolated from orange juice gave an unspecific cross-reaction with the 29 kDa peptide of the peel sample). The 65 kDa and the 46 kDa peptides appeared in the peptide samples of grapefruit and lemon juice, but the antibodies did not give a positive reaction with that of orange juice and all citrus peel extracts studied. Two lemon juice specific peptides with molecular mass of 35 kDa and 28 kDa were isolated. The antibodies gave positive reaction with a single 35 kDa and 28 kDa bands of peptides one by one and did not react with any peptides of orange and grapefruit juices and peel extracts of the three citrus fruits studied. The 31 kDa peptide isolated from grapefruit peel appeared in all the three peel samples of citrus fruits. This peptide could not be found in grapefruit and lemon juice, but a pale peptide band was detected in orange juice. Detection of juice and peel peptides in citrus juice products The next step in this work was to investigate whether the antibodies can differentiate the juice and peel peptides in processed citrus fruits. Figure 1 shows the blots of antibodies developed against 82 kDa citrus juice and 35 kDa lemon juice peptide. On the blot to the left, the antibody of 82 kDa peptide gave a single positive band with the peptide sample prepared from laboratory-processed grapefruit juice and the commercial grapefruit juice products. One sample
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
+
+
28 kDa
31 kDa
+
+
46 kDa
juice
35 kDa
+
65 kDa
+
27 kDa
peel
grapefruit juice
+
+
24 kDa
peel
orange
82 kDa
juice
ofpeptide
molecular mass
peel
lemon
Table Π. Isolation of peptides from citrus fruits
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
500-fold 250-fold 250-fold 500-fold 250-fold 15000-fold
82 kDa
65 kDa
46 kDa
35 kDa
28 kDa
31 kDa
(+) low concentration
1000-fold
(+)
(+)
+
+
peel
orange
of antibody juice
27 kDa
ofpeptide
Polyclonal antibody Dilution
+
+
+
+
(+) +
juice
+
+
peel
+
peel
lemon
+
+
+
juice
grapefruit
Table III. Distribution of isolated peptides in various part of citrus fruits
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
32
Figure I. Western blots ofantibodies developed against 82 kDa juice peptide and 35 kDa lemon juice peptide. (I) molecular mass standards; (2) grapefruit juice; (3) commercial 40 % grapefruit juice nectar; (4)commercial 100% grapefruitjuice; (5) lemon juice; (6) commercial lemon juice concentrate I; (7) commercial lemon juice concentrate II
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
33
Figure 2. Western blots of antibody developed against 31 kDa peel peptide. (1) molecular mass standards; (2) grapefruit peel extract; (3) grapefruit juice; (4) commercial 40 % grapefruitjuice nectar; (5) commercial 100% grapefruitjuice; (6) lemon peel extract; (7) lemon juice; (8) commercial lemon juice concentrate I; (9) commercial lemon juice concentrate II
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Downloaded by UNIV OF GUELPH LIBRARY on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch003
34 was a 100 % grapefruit juice, and the other one was 40 % red grapefruit nectarfromthe market. On the blot to the right, the antibody of 35 kDa peptide gave a positive reaction band with laboratory lemon juice and a pale, single positive band with the 35 kDa peptide of commercial lemon juice concentrates. The peptide bands were less intensive in processed lemon juice concentrate than in laboratory-processed lemon juice sample. Figure 2 shows the immune reaction of the antibody developed against 31 kDa peel peptide. The commercial citrusfruitssamples separated on the blots were the same as those in Figure 1. On the blot to the left, one of the commercial grapefruit juice products declared as 100% grapefruit juice did not give a positive reaction with this antibody while a pale, but well visible band appeared in the peptide sample of the 40% red grapefruit juice nectar. (On the box of the grapefruit nectar it was indicated that the nectar containedfruitflesh as well). On the blot to theright,a single positive reaction band can be observed with the lemon peel peptides prepared in the laboratory. No cross reactions occurred with the laboratory lemon juice sample. One of the juice concentrates did not give a positive reaction with peel antibody while a pale, but well visible band appeared in the peptide sample of one lemon juice concentrate. The results showed that the antibodies could detect the juice and peel peptides (or peptides originatingfromby-products) in commercial citrus juice products. In conclusion the antibody developed against peel peptide can be used to detect the manipulation of commercial citrus juice products by peel extract or other by-products. Antibodies developed against lemon juice peptides can be used to determine the authenticity of lemon juice products. Developing of a quantitative (ELISA) method for determining the juice content and the ratio of possible peel contamination in commercial citrus juice beverages using antibodies raised against these characteristic peptides is intended.
Acknowledgement The National Scientific Research Fund (OTKA) under Grant T029281 has supported this work.
References 1. 2. 3.
Laemmli U. K. Nature 1970, 227, 680-685. Towbin, H., Staehelin T., and Gordon J. Proc. Nat. Acad. Sci. U.S.A. 1979, 76, 4350-4354. Sass-Kiss, Α.; and Sass, M. J. Agric. Food Chem. 2000, 48, 40274031.
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
